The ability to prepare large amounts of nucleic acid molecules is requisite to a number of protocols in molecular biology, as well as a basic requirement in numerous downstream uses in biotechnology and clinical research. For example, amplified nucleic acid molecules are often used in cloning experiments, DNA sequencing reactions, restriction digestion reactions, and subsequent ligation reactions, and these uses are all, or to some extent, dependent on the quality and quantity of the starting DNA material. As such, there has been, and continues to be, a need for reliable methods for preparing large amounts of quality, sequence-specific nucleic acid molecules.
In addition, the ability to detect and/or quantify nucleic acid molecules from a mixed starting material is useful in a number of clinical, industrial and basic research applications. For example, sensitive and accurate detection and quantification of viral nucleic acid sequences in a patient sample is helpful in a clinical setting for accurate diagnosis and subsequent treatment of a patient. Such detection and quantification processes generally require amplification of one or more target nucleic acid molecules present in the starting material. As such, there has been, and continues to be, a need for facilitating the detection and quantification of target nucleic acid sequences from a starting material, which again requires reliable methods for preparing large amounts of quality, sequence-specific nucleic acid molecules.
The predominant approach for amplifying nucleic acid is via the polymerase chain reaction (PCR). PCR is a convenient in vitro amplification process useful in the exponential increase of template nucleic acid. One of the more critical facets of a successful PCR reaction is primer design, requiring specific primers that hybridize to the target template sequence. However, there is a relatively narrow range of reaction conditions (temperature, ion concentration, denaturing agents, etc.) where a primer will specifically anneal to its complementary target. Moreover, even with optimal primer selection, many PCR reactions produce little or no product due to non-specific amplification and/or primer aggregation.
Primer aggregation typically results from the extension of one primer off of the other primer in the PCR reaction, i.e., one primer acts as a template for the other primer; this process occurs even though no stable annealing of the primer is accomplished. Typically, as primers begin to form aggregates within a PCR reaction, the aggregates become valid templates for efficient PCR amplification, since the primer aggregates contain both primer annealing sites. In this manner, primer aggregation has a persistent and detrimental effect on PCR reactions, as each primer-aggregate acts as a template for additional rounds of non-specific amplification. Since the generation of primer aggregation is a major problem at low temperatures during the amplification reaction, i.e., prior to thermocycling, primer aggregation has typically been addressed using “hot start” technologies. Additional technologies for addressing this problem, however, are needed, as even “hot start” technologies are only partially successful in their approach to limiting primer aggregation. In addition, even small increases in non-specific amplification can lead to significant losses in sensitivity and specificity during a PCR or other like nucleic acid amplification reaction.
As such, there is a continuing need in the art for improvement of PCR techniques and compositions that allow for a reduction in primer aggregation during nucleic acid amplification reactions, and in particular PCR.